Influenza A virus is a major human respiratory pathogen that causes seasonal epidemics and occasional pandemics. The single-stranded negative-sense influenza virus genome consists of 8 segments, each flanked by short conserved elements at their termini, which form the viral promoter that is recognized by the viral RNA-dependent RNA polymerase (vPOL). Mutations in vPOL and factors that affect vPOL activity can cause adaptation of avian viruses to the human host. vPOL is essential for viral biogenesis because of its central role in transcription and replication of the viral genome. vPOL also severely impacts host transcription as it cleaves the capped ends of nascent cellular RNAs to prime viral transcription, a process known as Cap-snatch. The basis for these distinct vPOL activities, as well as the cellular factors that can affect its processivity in host cells still reain largely unknown. To address this, we developed reporter viruses that encode tagged vPOL subunits that do not adversely affect their infection potential. We will use these to characterize the vPOL interactome during productive infection, and to characterize the importance of epigenetic regulators and newly identified factors for influenza virulence (Aim 1). Moreover we will, for the first time, study the mechanism and impact of Cap-snatch by genome-wide profiling of the viral-cellular hybrid mRNAs using newly developed next-generation sequencing protocols (Aim 2). Finally, we will examine vPOL processivity, which may provide new insights into differential regulation of viral gene transcription during infection (Aim 3). Our proposal comprise the first comprehensive and integrated analysis of vPOL-host interactions at the genome, transcriptome and interactome level. Understanding these viral and cellular aspects in a human system will not only provide the first glimpse into early infection events, but also lead to new hypotheses for in vivo susceptibility in humans. Notably, the novel methodologies we developed for this proposal will be widely applicable to studying other types of viruses.